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Updated: Dec 8, 2025
Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1β in Human Monocyte-derived Dendritic Cells
Published on: May 22, 2014
MicroRNA-223 negatively regulates LPS-induced inflammatory responses by targeting NLRP3 in human dental pulp
1State Key Laboratory of Military Stomatology & National Clinical Research Center for Oral Diseases & Shaanxi Key Laboratory of Stomatology, Department of Operative Dentistry & Endodontics, School of Stomatology, Fourth Military Medical University, Xi'an, China.
Aim:
To investigate the effect of miR-223 on NLRP3, subsequently regulating the production of the NLRP3/CASP1 inflammasome pathway-mediated proinflammatory cytokines IL-1β and IL-18 in human dental pulp fibroblasts (HDPFs).
Methodology:
Human dental pulp tissue (HDPT) and HDPFs were obtained from impacted third molars. The miR-223 mimics and inhibitor or NLRP3 plasmid were used to upregulate or downregulate miR-223 or NLRP3 in HDPFs, respectively. Computational prediction via TargetScan 5.1 and a luciferase reporter assay was conducted to confirm target association. The mRNA and protein expression of NLRP3, caspase-1, IL-1β and IL-18 was determined by qRT-PCR and Western blotting, respectively. The release of IL-1β and IL-18 was analysed by ELISA. The significance of the differences between the experimental and the control groups was determined using one-way analysis of variance; P < 0.05 indicated statistical significance.
Results:
A decrease in miR-223 and an increase in NLRP3 in HDPT occurred during the transformation of reversible pulpitis into irreversible pulpitis compared to that in healthy pulp tissue (P < 0.05). The computational prediction and luciferase reporter assay confirmed that NLRP3 was a direct target of miR-223 in HDPFs. The miR-223 inhibitor further promoted ATP plus LPS-induced NLRP3/CASP1 inflammasome pathway activation compared to the ATP plus LPS-induced group (P < 0.05). In contrast, the miR-223 mimic significantly inhibited the NLRP3/CASP1 inflammasome pathway activation induced by ATP plus LPS compared to the ATP plus LPS-induced group (P < 0.05).
Conclusion:
MiR-223 served as a negative regulator involved in the control of the production and secretion of proinflammatory cytokines mediated by the NLRP3/CASP1 inflammasome pathway by targeting NLRP3. These data provide insight into the potential regulatory effects of miRNAs on the NLRP3 inflammasome, thus opening up novel potential therapeutic avenues for future endodontic treatment.
Insights
MicroRNA-223 (miR-223) negatively regulates the NLRP3 inflammasome pathway, reducing the production of inflammatory cytokines IL-1β and IL-18. This finding offers potential therapeutic strategies for endodontic treatments targeting pulpitis.
Area of Science:
- Molecular Biology
- Immunology
- Endodontics
Background:
- Pulpitis involves inflammation mediated by the NLRP3 inflammasome pathway.
- MicroRNAs (miRNAs) play crucial roles in regulating cellular processes, including inflammation.
Purpose of the Study:
- To investigate the regulatory role of miR-223 in the NLRP3/CASP1 inflammasome pathway in human dental pulp fibroblasts (HDPFs).
- To determine if miR-223 targets NLRP3 and influences the production of IL-1β and IL-18.
Main Methods:
- HDPFs and human dental pulp tissue were used.
- miR-223 mimics/inhibitors and NLRP3 plasmids modulated gene expression.
- Target validation was performed using computational prediction and luciferase assays.
- Gene and protein expression (NLRP3, caspase-1, IL-1β, IL-18) were assessed via qRT-PCR and Western blotting.
- Cytokine release was measured by ELISA.
Main Results:
- miR-223 levels decreased while NLRP3 increased in pulpitis compared to healthy tissue.
- NLRP3 was confirmed as a direct target of miR-223.
- miR-223 inhibition enhanced NLRP3/CASP1 inflammasome activation, while miR-223 mimicry inhibited it.
Conclusions:
- miR-223 acts as a negative regulator of the NLRP3/CASP1 inflammasome pathway by targeting NLRP3.
- This regulation impacts the production and secretion of IL-1β and IL-18.
- miRNAs, specifically miR-223, represent potential therapeutic targets for endodontic treatments.
